GSM7179124: kidney, healthy, replicate 1; Mus musculus; RNA-Seq
Kidneys were collected and then digested in 5mg/mL collagenase D (Roche Diagnostics) and 100μg/mL DNase I (Sigma-Aldrich), in Hanks balanced salt solution without Ca2+ and Mg2+ (Sigma-Aldrich) for 30 minutes at 37℃. Cells were erythrocyte lysed with NH4Cl and filtered with 40μM filter (BD Bioscience). Dead cells and debris were removed using Dead Cell Removal Kit (Miltenyi) leaving alive cells that were counted on a hemocytometer with 0.4% trypan blue (Sigma-Aldrich). Single-cell concentration was adjusted to between 700-1200 single cells/μL in 0.04% BSA/PBS. Cells were loaded onto a 10x Chromium Controller (10x Genomics) to perform single-cell partitioning and barcoding. scRNA-seq libraries were constructed using 10x Genomics Chromium Single Cell 3' V3.1 chemistry according to the manufacturer's instructions (10x Genomics). Sample quality was determined by CE integrity analysis by AATI Fragment Analyzer and fluorimetric quantitation by Qubit (Invitrogen).